Protective Effects of Heat-Killed Ruminococcus albus against β-Amyloid-Induced Apoptosis on SH-SY5Y Cells

A high level of β-amyloid (Aβ) in the brains of patients with Alzheimer’s disease (AD) generates reactive oxygen species that induce neuronal death and DNA damage. The interaction between the gut microbiota and brain health has attracted attention in recent years. Heat-killed Ruminococcus albus (hkRA) reportedly protects neurons against damage induced by oxidative stress. However, whether hkRA can inhibit Aβ-induced apoptosis and thus alleviate AD remains unclear. Hence, we aimed to evaluate the protective effects of hkRA against Aβ-induced apoptosis on the human neuroblastoma SH-SY5Y cell. HkRA treatment (108 cells/ml) significantly decreased the Aβ-induced cytotoxicity and DNA damage in the SH-SY5Y cells. It also showed a significant increase of the bax/bcl-2 ratio in the Aβ-treated SH-SY5Y cells. Moreover, hkRA treatment stimulated the expression of antioxidation-related genes HO-1, Nrf2, and PKC-δ and increased the expression of brain-derived neurotrophic factor (BDNF). Meanwhile, it significantly decreased the activity of caspase-3 and protein expression of cleaved caspase-3 in the Aβ-treated SH-SY5Y cells. Additionally, the protein levels of mitochondrial and cytosolic cytochrome c increased and decreased, respectively, in the cells. These results suggest that hkRA protects human neuroblastoma cells from Aβ-induced apoptosis and oxidative stress. Thus, hkRA may be developed into a health-promoting paraprobiotic (the inactivated microbial cells of probiotics) for patients with AD.

and rivastigmine, and memantine, a N-methyl-D-aspartate receptor antagonist [17].AD is also alleviated by antiinflammatory drugs, estrogen, antioxidants, Aβ protein deposition, and aggregation inhibitors [18].However, none of these therapeutics have been used for the complete treatment of AD.
Recently, clinical trials have shown that probiotics are promising for AD prevention [19,20].Live and inactivated probiotics exert similar health benefits [21][22][23].Nevertheless, inactivated probiotics (known as paraprobiotics) have some advantages over live probiotics, such as no risk of infection in compromised individuals, stability, and ease of production and handling (transportation and storage).Heat-killed probiotics are currently receiving attention for their health-promoting effects, but only limited information is available [24,25].Ruminococcus albus is a gram-positive intestinal bacterium that is primarily cellulolytic.In our previous study, we found that substances secreted from heat-killed R. albus (hkRA)-treated intestinal epithelial Caco-2 cells increase the proliferation of neuronal SH-SY5Y cells and protect them against H 2 O 2 -induced oxidative stress by activating the expression of brain-derived neurotrophic factor (BDNF), serum response factor, and cyclin-dependent kinase 2 [26].In addition, hkRA protects neurons from oxidative damage induced by sodium arsenate in rats.Considering the neuronal cell death in AD caused by oxidative stress and Aβ protein accumulation [2,6] and the protective potency of hkRA on oxidative stress-induced neuronal damage, we hypothesized that hkRA itself and not its metabolites can alleviate AD by inhibiting Aβ-induced neurotoxicity.In this study, we aimed to investigate the direct protective effects of hkRA against Aβ-induced apoptosis not through the gut-brain axis, thereby preventing DNA damage, and elucidate the underlying mechanisms in SH-SY5Y cells.

Preparation of hkRA
Ruminococcus albus KCTC 15045 was obtained from the Korean Collection Type Culture (Republic of Korea).The bacterium was cultured under anaerobic conditions in modified DSMZ 453 medium (Leibniz Institute, Germany) at 37 o C. R. albus was harvested by centrifugation at 3,000 ×g for 5 min and washed with phosphatebuffered saline (PBS).After centrifugation, the pelleted cells were resuspended in PBS by gentle vortexing and were heat killed at 100 o C for 10 min and then stored in a -80 o C deep freezer until further use.

Preparation of Aβ 25-35 Aggregates
Aβ 25-35 (Sigma-Aldrich, USA) (1 mM) in distilled water stored at -20 o C until use.For aggregation of Aβ 25-35 to increase cytotoxicity, it was incubated in a 37 o C water bath for 72 h [27].In all experiments, Aβ 25-35 was treated to the final concentration of 10 μM.

Cell Culture
The human neuroblastoma SH-SY5Y cell line was purchased from the Korean Cell Line Bank (Republic of Korea ).SH-SY5Y cells were cultured in DMEM supplemented with high glucose containing 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/ml each) (HyClone, USA) at 37 o C in 5% CO 2 .All experiments were performed 24 h after seeding the cells onto the plates.SH-SY5Y cells were pretreated with various concentrations (10 6 , 10 7 , and 10 8 cells/ml) of hkRA for 30 min and then exposed to 10 μM Aβ 25-35 dissolved in distilled water for 24 h.Only cell passages under 30 were used in all experiments.

Soft Agar Colony Formation Assay
A soft agar colony formation assay (clonogenic assay) was performed to evaluate the effect of hkRA pretreatment on the proliferation of Aβ 25-35 -treated SH-SY5Y cells.For the base layer, a mixture composed of 0.5% agar, DMEM, and 10% FBS was prepared.In a 6-well plate, 1 ml of the mixture was added to each well and allowed to solidify for 5 min.For the top layer, 5 × 10 3 cells/well were mixed with 0.35% low-melting agarose (Sigma-Aldrich) and Aβ 25-35 (10 μM).Various concentrations (10 6 , 10 7 , and 10 8 cells/ml) of hkRA were also added to the top-layer agar.The cells were incubated at 37 o C in 5% CO2 for 2 weeks.The plates were fixed with formaldehyde and then stained with crystal violet for 30 min.

Comet Assay
DNA damage was evaluated using the comet assay, which involves measuring the migration distance of the nuclei through electrophoresis of the DNA of damaged cells.SH-SY5Y cells were harvested by centrifugation at 400 ×g for 5 min.The cells were mixed with 0.75% low-melting point agarose and immediately spread on frosted microscope slides precoated with 0.75% normal agarose.The slides were covered with coverslips and incubated to allow gel solidification for 10 min at 4 o C.Then, the slides were immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% Triton X-100, pH 10) for 1 h at 4 o C.After lysis, the slides were placed in electrophoresis buffer overnight and then electrophoresed at 60 V for 20 min.After electrophoresis, the slides were neutralized with 0.4 M Tris, pH 7.5.The gels were stained with propidium iodide (Sigma-Aldrich) and dried overnight at room temperature.Images were obtained using a confocal microscope (C1 PLUS; Nikon, Japan).The tail moment (tail length × DNA in tail (%)) was calculated using CaspLab software (version 1.2.3.beta 2) (CASPlab, Wroclaw, Poland).

Quantitative Real-Time Polymerase Chain Reaction (qPCR)
Total RNA was extracted using AccuZol Total RNA Extraction Reagent (Bioneer, Korea) in accordance with the manufacturer's protocol.The PCR primers were purchased from Bioneer (Table 1).The PCR conditions were as follows: 95 o C for 10 min, followed by 40 cycles at 95 o C for 15 s, 60 o C for 15 s, and 72 o C for 20 s.Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was used as the internal control gene.Normalized expression change was expressed as 2 -ΔΔCt (The control GAPDH was set to 1).

Caspase-3 Activity
Caspase-3 activity was measured using a caspase-3 colorimetric assay kit (BioVision, USA) in accordance with the manufacturer's instructions.Briefly, SH-SY5Y cells were lysed in a caspase-3 sample lysis buffer and then incubated on ice for 10 min.After 10 min, the lysate was centrifuged at 10,000 ×g at 4 o C for 1 min.The supernatant was incubated with DEVD-pNA substrate (200 μM final concentration) at 37 o C for 1 h, and the absorbance was obtained at 405 nm using the SpectraMax 340PC384 plate reader (Molecular Devices, USA).

Western Blot
Proteins were extracted using the Pro-prep Protein Extract solution (Intron, Republic of Korea) in accordance with the manufacturer's protocol.Mitochondrial and cytosolic protein fractions were prepared using a mitochondria/cytosol fractionation kit (Biovision).In brief, cells were incubated in the Pro-prep solution at -20 o C for 30 min and then centrifuged at 13,000 ×g for 5 min at 4 o C. The supernatants were collected, and the protein concentrations were measured using Bradford assay.Equal amounts (30 μg) of proteins for each sample were separated through 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, USA) using a Trans-Blot semi-dry transfer cell (Bio-Rad, USA).The membranes were blocked with 5% (w/v) skim milk (Bio-Rad) in PBS-T (PBS containing 0.05% Tween-20) at room temperature for 1 h, washed three times with PBS-T for 10 min, and then incubated with primary antibodies against β-actin (1:5,000 dilution, MA5-15739; Thermo Fisher Scientific), caspase-3 p11 (1:500 dilution, sc-271759; Santa Cruz Biotechnology, USA), and cytochrome c (1:500 dilution, sc-13156; Santa Cruz Biotechnology) for 1 h at room temperature.After being washed three times with PBS-T for 10 min each, the membranes were incubated with goat anti-mouse IgG (H+L) horseradish peroxidase-conjugated secondary antibodies (1: 20,000 dilution; NCI1430KR, Thermo Fisher Scientific) for anti-β-actin, anti-caspase-3 p11, and anti-cytochrome C for 1 h at room temperature.Proteins were detected using the SuperSignal West Femto Maximum Sensitivity Substrate Kit (Thermo Fisher Scientific).Blot images were analyzed using the FluorChem E imaging system (ProteinSimple, USA).Protein density was quantified using ImageJ software (Sofomic, Barcelona, Spain).

Statistical Analysis
All statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test using SPSS version 24.0.Results are expressed as the mean ± SD of three independent experiments performed in triplicate.A p-value of less than 0.05 was considered statistically significant.

Effects of hkRA Pretreatment on the Viability of Aβ 25-35 -Treated SH-SY5Y Cells
Cell viability was not significantly different in all hkRA-treated SH-SY5Y cells, except for the 10 8 cells/ml of hkRA-treated cells.The hkRA concentration of 10 8 cells/ml significantly increased the viability of the SH-SY5Y cells by 130.97% ± 1.66% compared with the NC (100%) (Fig. 1A).Therefore, hkRA at concentrations of 10 6 , 10 7 , and 10 8 cells/ml was used in subsequent experiments in this study.The viability of the SH-SY5Y cells treated only with Aβ 25-35 alone significantly decreased by 53.31% ± 0.94% compared with that of the NC (Fig. 1B).Although pretreatment with 10 8 cells/ml hkRA only significantly (p = 0.001) protected the cells against Aβ 25-35 toxicity, pretreatment with 10 6 or 10 7 cells/ml hkRA also increased cell viability.
Aβ 25-35 induces neuronal apoptosis and is used as a substitute for Aβ 1-42 because it immediately forms a βpleated sheet structure similar to Aβ 1-42 and has high toxicity [27].In addition, the 25-35 region of Aβ contributes Table 1.Primer sequences used for qPCR.

Gene
Forward (5-3) Reverse (5-3) to the aggregation of amyloid fibrils and neurotoxicity in peptides.We found that Aβ 25-35 was highly toxic to the SH-SY5Y cells.Without Aβ 25-35 treatment, a significant difference in viability was not found between the cells pretreated with 10 2 -10 7 cells/ml hkRA and PBS.Moreover, the cells pretreated with 10 8 cells/ml hkRA showed higher viability than those treated with the control, confirming the safety of hkRA at the concentrations used in this study.Treatment with Aβ 25-35 significantly decreased the viability of the cells, and although this effect was not significant, the viability in the hkRA pre-treated-cells decreased as well compared with the NC.Nevertheless, pretreatment with hkRA, especially 10 8 cells/ml hkRA, showed a significant protective effect against the Aβ 25-35induced cytotoxicity.

Effect of hkRA Pretreatment on the Proliferation of Aβ 25-35 -Treated SH-SY5Y Cells
The effect of hkRA pretreatment on the proliferation of Aβ 25-35 -treated SH-SY5Y cells was evaluated through soft agar colony formation assay.The colony formation of the Aβ 25-35 only-treated cells (24.30% ± 7.87%) was significantly lower than that of the cells treated with the control (100%) (Fig. 2).Pretreatment with hkRA showed a significant dose-dependent increase in the colony formation of the Aβ 25-35 -treated SH-SY5Y cells.The results suggested that hkRA protected the proliferation of cells under Aβ 25-35 treatment.

Fig. 2. Proliferation of SH-SY5Y cells pretreated with heat-killed Ruminococcus albus (hkRA) before βamyloid (Aβ) treatment.
Representative colony formation images of hkRA-pretreated SH-SY5Y on soft agar plates (A) and the colonies were counted (B).Data are expressed as the mean ± SD of three independent experiments in triplicate.One-way ANOVA/Tukey's honestly significant difference analysis was performed.

Effect of hkRA on the Expression of Apoptosis-and Antioxidation-Related Genes in Aβ 25-35 -Treated SH-SY5Y Cells
The degree of apoptosis was determined by calculating the expression ratios between bax and bcl-2, a proapoptotic gene and an anti-apoptotic gene, respectively [28].The expression ratio of bax/bcl-2 significantly (p < 0.000) increased by 3.3-fold in the cells treated only with Aβ 25-35 compared with the NC (Fig. 4A).However, this ratio decreased by 0.37-, 0.32-, and 0.23-fold in the cells pretreated with 10 6 , 10 7 , and 10 8 cells/ml hkRA, respectively, compared with the cells treated with Aβ 25-35 alone.Aside from markedly decreasing the distance of DNA fragment migration, hkRA pretreatment also decreased the bax/bcl-2 ratio, confirming its protective effect against Aβ 25-35 -induced apoptosis in the SH-SY5Y cells.
The expression level of BDNF was significantly lower in the cells treated only with Aβ 25-35 than in the NC cells; however, it was significantly higher in the cells pretreated with all concentrations of hkRA than in those treated only with Aβ 25-35 (Fig. 4B).Interestingly, the expression of BDNF was significantly higher in the cells pretreated with all concentrations of hkRA than in those treated with the control.BDNF is a brain growth hormone that stimulates the growth and division of new neurons and aids in their survival [29].BDNF levels decrease with aging, accelerating brain damage [30].Thus, BDNF may be a key factor in delaying brain aging while also being an important target in improving degenerative brain diseases.Pretreatment with hkRA notably increased BDNF expression in the Aβ 25-35 -treated SH-SY5Y cells, suggesting that hkRA could be a neuroprotective substance.
Nerve damage can also be induced by ROS in apoptotic cells stimulated by Aβ [6].Therefore, the expression of genes related to ROS inhibition may also be an important indicator of neuroprotective effects in Aβ-treated cells.Transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a major regulator of the expression of antioxidant proteins that maintain intracellular antioxidant activity and performs an important function in maintaining intracellular redox homeostasis.Nrf2 induces the transcription of various cytoprotective genes that prevent the deleterious effects of oxidative stress and toxicants.Heme oxygenase-1 (HO-1) is a typical antioxidant enzyme that responds to oxidative stress and is expressed through the PKC-δ/Nrf2/ARE signaling pathway in SH- SY5Y cells [7,31].In this study, the expression levels of antioxidation-related genes PKC-δ, Nrf-2, and HO-1 were significantly higher in the cells pretreated with 10 8 cells/ml hkRA than in those treated with Aβ 25-35 alone (Fig. 4C-4E).This result suggested that hkRA showed a neuroprotective effect against oxidative stress by activating the PKC-δ/Nrf2/ARE signaling pathway in SH-SY5Y cells.

Inhibitory Effects of hkRA on Mitochondrial Cytochrome c Release and Caspase-3 Activity in Aβ 25-35 -Treated SH-SY5Y Cells
Cytochrome c release was measured through western blot to investigate the inhibitory effect of hkRA on cytochrome c release from the mitochondria to the cytoplasm in the Aβ 25-35 -treated SH-SY5Y cells (Fig. 5A).In the cells treated with Aβ 25-35 alone, the protein level of cytochrome c in the mitochondria significantly decreased Oxidative stress and mitochondrial dysfunction are important causes of Aβ-mediated neurotoxicity [32].Aβ accumulates in the mitochondria of AD patients' brains, which reveal mitochondrial structural abnormalities [33], and results in the release of cytochrome c from the mitochondria to the cytoplasm [34].Cytochrome c is released from the mitochondrial inner membrane and binds to Apaf-1 (apoptosis protease-activating factor), which then recruits procaspase-9 to form an apoptosome that cleaves procaspase-9 to activate caspase-9.Activated caspase-9 stimulates caspase-3, a marker of apoptosis.The level of cleaved caspase-3, a terminal enzyme in apoptosis, is significantly correlated with cancer progression [35].The level of cleaved caspase-3 decreases as amyloid-β-induced neuronal damage is ameliorated by the overexpression of fibroblast growth factor 13 [36].In the present study, hkRA pretreatment significantly reduced cytosolic cytochrome c level in the Aβ 25-35 -treated SH-SY5Y cells.In addition, it significantly reduced the activity of caspase-3 and the protein expression of cleaved caspase-3 in the Aβ 25-35 -treated SH-SY5Y cells.These results indicated that hkRA protected the cells from Aβ 25-35induced apoptosis by decreasing cytochrome c release from the mitochondria to the cytoplasm, which decreased the generation of cleaved caspase-3, the active form of caspase that plays an important role in apoptosis [37].
R. albus, an important cellulolytic bacterium in the intestine, is a next-generation probiotic, and healthy individuals possess higher loads of R. albus than patients with Crohn's disease or immunodeficiency disorders [38,39].hkRA has health-promoting effects similar to those of live bacterial cells [26].In the present study, hkRA pretreatment protected neurons from Aβ-induced neurotoxicity, suggesting that hkRA may be developed as a paraprobiotic for patients with AD.Additionally, further extensive study on the identification of the active In conclusion, hkRA prevents neuronal damage from Aβ 25-35 -induced oxidative stress and protects neurons from Aβ 25-35 -induced apoptosis.hkRA also upregulates the expression of BDNF, a major factor in the protection and restoration of neurons.Thus, based on the results obtained from this in vitro study using the SH-SY5Y cell model, further in vivo studies are needed to justify if hkRA passes directly through the blood-brain barrier, and its improvement effect on Aβ 25-35 -induced neuronal diseases, such as AD, by protecting neurons from damage and promoting their growth.

Author Contributions
YHL conceptualized and designed the study.SC performed the experiments, and SC and MA performed the statistical analyses and drafted the manuscript.YHL reviewed and edited the manuscript.

Fig. 5 .
Fig. 5. Inhibitory effect of heat-killed Ruminococcus albus (hkRA) on cytochrome c release in Aβ 25-35 -treated SH-SY5Y cells.Protein levels of cytochrome c were determined using western blot (A) and cytochrome c levels were quantified in the mitochondria (B) and cytosol (C).Data are expressed as the means±SD of three independent experiments.One-way ANOVA/Tukey's honestly significant difference analysis was performed.

Fig. 6 .
Fig. 6.Effects of heat-killed Ruminococcus albus (hkRA) on the protein expression of cleaved caspase-3 and activity of caspase in Aβ 25-35 -treated SH-SY5Y cells.Protein expression of cleaved caspase-3 was determined using western blot (A) and quantified (B).Caspase activity was measured using a colorimetric assay (C).Data are shown as the means±SD of three independent experiments.One-way ANOVA/Tukey's honestly significant difference analysis was performed.